斑节对虾肝胰腺全长cDNA表达文库的构建

Construction of full-length cDNA expression library of hepatopancreas of Penaeus monodon

采用PolyATtract1000系统提取完整的斑节对虾肝胰腺mRNA,ZAPExpresscDNA合成试剂盒合成具有EcoRⅠ和XhoⅠ末端的双链cDNA.cDNA与经EcoRⅠ和XhoⅠ预消化的λZAPExpress载体连接,再经GigapackⅢGold蛋白体外包装和感染E.coli菌株XL1-BlueMRF′,成功构建了库容量为7 7×105,重组率高达99 1%的cDNA表达文库.将初级文库进行扩增,并在滴定扩增文库时随机挑取9个噬菌斑,在辅助噬菌体ExAssist的作用下通过体内切除形成phagemid,再用EcoRⅠ和XhoⅠ双酶切获得插入cDNA片段,其长度最长达1 6kb.我们还用PCR法从库中扩增出完整的肌动蛋白编码区cDNA(约1 2kb).以上结果说明此表达文库含有斑节对虾肝胰腺全长cDNA片段,因此本文库为对虾基因的克隆、表达及其结构和功能的研究奠定了基础.

mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyATtract System1000 Kit. By using mRNA as template, double-strand cDNA with EcoRⅠ/XhoⅠends was synthesized by using a ZAP expression cDNA Synthesis Kit. The cDNA was inserted into the lambda ZAP expression vector predigested with EcoRⅠ/XhoⅠ, and the recombinant DNA was in vitro packaged into lambda phage with GigapackⅢ Gold packaging extracts. These recombinant phages were then used to transfect E. coli XL1-Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2×10~5 pfu in capacity and its recombination ratio is higher than 99%. The size of the inserted cDNAs was determined by EcoRⅠ/XhoⅠ digestion of 9 phagemids prepared by in vivo excision of plaques selected randomly from amplified cDNA library. The longest inserted cDNA is about 1.6 kb in length. The complete sequence (about 1.2 kb) of actin cDNA amplified from the library by PCR reveals that this library contains full-length cDNAs of shrimp Penaeus monodon hepatopancreas and is available for screening and expression of shrimp genes.